Purified Plasmodium and vaccine composition

ABSTRACT

Disclosed are substantially purified  Plasmodium  sporozoites and preparations of  Plasmodium  sporozoites substantially separated from attendant non-sporozoite material, where the preparations of  Plasmodium  sporozoites have increasing levels of purity. Vaccines and pharmaceutical compositions comprising purified  Plasmodium  sporozoites are likewise provided. Methods of purifying preparations of  Plasmodium  sporozoites are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This invention was developed in part under the auspices of an agreementbetween Sanaria, Inc. (Dr. Stephen L. Hoffman—CEO) and ProteinPotential, Inc. (Dr. Kim Lee Sim—President) and is entitled to thebenefits of the CREATE Act of 2004. This application is a divisionalapplication of U.S. Ser. No. 12/870,102, filed Aug. 27, 2010, nowgranted U.S. Pat. No. 8,367,810, which is a divisional application ofU.S. Ser. No. 12/684,863, filed Jan. 8, 2010, now granted U.S. Pat. No.8,043,625, which claims priority to U.S. Ser. No. 61/202,001, filed Jan.16, 2009, each of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This application relates to purification of eukaryotic pathogens andparasites, particularly, Plasmodium sporozoite-stage parasites. Moreparticularly it relates to substantially pure parasites and methods ofpreparing and using them. The application also relates to vaccine andpharmaceutical compositions of purified sporozoite stage Plasmodiumparasites, both attenuated and non-attenuated, and methods of using thecompositions in vaccines and other preparations to prevent malaria andother diseases, treat diseases, and as a means to infect volunteers inthe testing of malaria vaccines and drugs.

2. Background Art

Malaria is a disease that is estimated to affect 300-500 million peopleand kills 1-3 million individuals annually. It also has an enormouseconomic impact on people in the developing world, especially those insub-Saharan Africa. Plasmodium falciparum accounts for the majority ofdeaths from malaria in the world. The World Tourist Organizationreported that of the nearly 700 million international tourist arrivalsrecorded worldwide in 2000, approximately 9 million were to West,Central or East Africa, 37 million were to South-East Asia, 6 million toSouth Asia and 10 million to Oceania. It is estimated that more than30,000 travelers from North America, Europe, and Japan contract malariaper year. For more than 100 years during every military campaignconducted where malaria was transmitted, U.S. forces have had morecasualties from malaria than from hostile fire. An estimated 12,000,000person days were lost during World War II and 1.2 million during theVietnam conflict due to malaria.

Transmission of the Plasmodium parasite occurs through the bite andfeeding of infected female Anopheles mosquitoes which are active fromdusk to dawn. Plasmodium, at the sporozoite stage of development,migrate from the bite site to the liver, primarily via the blood stream,where they multiply within hepatocytes, producing, in the case of P.falciparum, about 10,000-40,000 progeny per infected cell. These liverstage parasites express some proteins which are not expressed at thesporozoite stage. At this stage, parasites re-enter the blood stream asmerozoites, expressing some proteins which are different from thoseexpressed during the sporozoite and early hepatic stages, and invadeerythrocytes, where additional multiplication increases parasite numbersby approximately 10 to 20 fold every 48 hours. Unlike the five to tenday development in the liver, which does not induce any symptoms orsigns of malaria, untreated blood stage infection causes hemolysis,shaking chills, high fevers, and prostration. In the case of P.falciparum, the most dangerous of the four major species of Plasmodiumthat cause human disease (P. vivax, P. malariae, and P. ovale [P.knowlesi can also cause human disease]), the disease is complicated bydisruption of microcirculatory blood flow and metabolic changes in vitalorgans such as the brain, kidneys and lungs, frequently leading to deathif not urgently treated.

An effective vaccine against P. falciparum malaria remains one of thegreat challenges of medicine. Despite over one hundred years of effort,hundreds of millions of dollars in research, lifelong sacrifice fromdedicated physicians and scientists, and many promising experimentalvaccines, there is no marketed vaccine to alleviate one of the greatinfectious scourges of humanity.

A generation ago, public health initiatives employing chloroquine, DDTand vector control programs seemed poised to consign falciparum malariato insignificance as a worldwide menace. The lack of an effectivevaccine complicated these efforts, but sustainable control seemedimminent.

The promise of impending success was short-lived and the reasons forfailure were multi-factorial. The parasites grew increasingly resistantto highly effective and affordable anti-malarial medications, vectorcontrol measures lapsed, and trans-migration, war and economicdisruption became increasingly more common in endemic areas of thedeveloping world. As a result, P. falciparum malaria resurged, annuallyplacing at least 2.5 billion humans at risk, causing 300-900 millioninfections, and killing 1-3 million people. Of the many social,economic, environmental and political problems that afflict thedeveloping world, P. falciparum malaria is increasingly seen as both aroot cause and cruel result of these inequities, and is a singularimpediment to solving these complex problems. Controlling P. falciparummalaria in the developing world may not be possible without an effectivevaccine. In practice, given social, political and economic realities, webelieve that a vaccine may be an essential component of a sustainablecontrol program, and will be required for a global eradication campaign.

During the last 25 years most research effort has been spent onidentifying the antigenic subunits of the parasite which conferimmunity—unfortunately, with less than satisfying results. This effortand the attendant difficulties in developing a suitable vaccine havebeen described (Nussenzweig V., F. and R. S. Nussenzweig, Adv. Immunol.,(1989) 45: 283-334; Hoffman S. L. et al. In: Hoffman S. L., ed. MalariaVaccine Development: A Multi-Immune Response Approach (1996) Washington,D.C.: ASM Press, pp. 35-75; Hoffman S. L. and L. H. Miller, In: HoffmanS. L., ed. Malaria Vaccine Development: a Multi-Immune ResponseApproach. (1996) Washington, D.C.: ASM Press, pp. 1-13; Epstein, J. E.et al, Curr. Opin. Mol. Ther. (2007) 9:12-24; Richie, T. L. & A. Saul,Nature, (2002) 415:694-701).

There are continuing efforts to produce subunit malaria vaccines.Typical of such attempts, Paoletti et al. (U.S. Pat. No. 5,766,597,issued Jun. 16, 1998) disclose a recombinant poxvirus containing DNAfrom Plasmodium coding for one or more circumsporozoite proteins,including an embodiment termed NYVAC-Pf7, possibly useful as a potentialmalaria vaccine. Subsequent testing of this construct proved to bedisappointing (Ockenhouse, C. F. et al. J. Infect. Dis. (1998) 177:1664-73).

Similarly, another candidate subunit circumsporozoite vaccine wasproposed and identified as RTS, S/AS02A (Stoute J. A. et al. J. Infect.Dis. (1998) 178: 1139-44). The results of the first Phase 2b field trialof this vaccine in one-four year old children in Mozambique was reported(Alonso, P. L. et al. Lancet (2004) 364:1411-1420; Alonso, P. L. et al.Lancet (2005) 366:2012-2018; Epstein, J. E. et al, Supra; Richie, T. L.,F. & A. Saul, Supra), as were the results of other Phase 2b field trialsin infants (Aponte, J. J. et al. (2007) The Lancet 370:1543-1551; Bejon,P. et al (2008) NEJM 359:2521-32; Abdullah, S et at (2008) NEJM359:2533-44. The vaccine has demonstrated modest protective efficacy.

On the other hand, the demonstration of the effectiveness of wholeparasite, radiation attenuated sporozoites (delivered to human hosts bymosquito exposure and to animal hosts by intravenous (i.v.) inoculation)in conferring high levels of protective immunity when recipients aresubsequently challenged with pathogenic parasites (most importantlyattenuated Plasmodium falciparum to human hosts) was an early milestonein the quest for a suitable vaccine (Hoffman S. L. et al., J. Infect.Dis. (2002) 185: 1155-64). Eventually, this led to efforts to explorethe technical hurdles which present themselves in transforming theseearlier observations into a practical vaccine approach comprisingaseptic attenuated sporozoites (Luke, T. C. & S. L. Hoffman, J. ExP.Biol., (2003) 206:3803-3808; Hoffman, S. L., and T. C. Luke U.S. Pat.No. 7,229,627 and U.S. Publication 2005/0208078). It has also led toincreased interest generally in the utility of vaccines utilizingattenuated sporozoites (Menard, R., Nature 2005) 433:113-114; Waters, A.P. et al. Science. (2005) 307:528-530; Wykes, M. F. & M. F. Good, Int.J. Parasitol. (2007) 37:705-712; Renia, L. et al, Expert Rev. Vaccines,(2006) 5:473-481; Epstein, J. E. et al, Supra).

Other modes of attenuation have also been demonstrated. For example, Itwas shown that attenuated sporozoites resulting from gene alteration ofPlasmodium berghei protects mice against P. berghei malaria (Kappe etal. U.S. Pat. No. 7,122,179; Mueller et al. Nature (2005); Mueller, etal. PNAS (2005); van Dijk et al. PNAS (2005) 102:12194-12199); Waters,U.S. Pat. No. 7,550,138; Labaied et al. Infect. And Immun. (2007).Recently genetic attenuation of the sporozoites of P. falciparum has bedisclosed (van Schaijk et al. PLoS ONE (2008) 3:e3549); VanBuskirk etal. PNAS.

Similarly, chemical attenuation of Plasmodium has be described (Purcellet al. Infect. Immun. (2008)76:1193-99; Purcell et al Vaccine (2008)26:4880-84).

Others have also described methods of culturing unpurified preparationsof sporozoites and inducing parasite differentiation to axenic liverstages (Kappe et al. US Pub. 2005/0233435).

The studies discussed above set forth certain limitations. For example,while sporozoites delivered to human hosts by the bite of a mosquitogenerate an effective immune response against malaria, such a method ofdelivery is clearly not a practical method for vaccinating a populationin need of protection against malaria. Additional studies in mice,referred to above, have suggested that the delivery of attenuatedsporozoites to mice intravenously are also effective, whereas othermeans of delivery (e.g., intramuscular) in comparison, are not. Anintravenous delivery method, however, is also not practical if amalarial vaccine is to be delivered to numerous individuals (includingchildren and the elderly). Intravenous delivery has increased risks,increased costs, and patients are far less likely to agree to bevaccinated using such a method. Thus, there was a need in the art toprovide an effective malarial vaccine that provides protective immunity,where the vaccine can be administered by a variety of methods.

With regard to considerations for human vaccines, the purity of theimmunogen and the presence or absence of non-specific, attendantmaterial which may be immunogenic or toxic are further essential issueswhich have not previously been resolved. Isolated attenuated sporozoitepreparations, as used in studies discussed above, contain contaminatingand other attendant material. There was a need in the art to developsporozoite-based vaccines, particularly in humans, that employaseptically prepared, sterile, purified preparations of sporozoites foruse in vaccine compositions. Such aseptically prepared, purifiedpreparations of sporozoites may also be more effective than non-purifiedpreparations when such preparations are administered by non-intravenousdelivery methods. (e.g., intramuscular, intraperitoneal, intradermal,epidermal, mucosal, submucosal, cutaneous, or subcutaneous).

BRIEF SUMMARY OF THE INVENTION

Disclosed are compositions of live, infectious, substantially purifiedsporozoites, particularly Plasmodium sporozoites—attenuated sporozoitesas well as pathogenic sporozoites. Also disclosed are methods of makinglive, infectious, substantially purified parasites and methods of usingcompositions of substantially purified attenuated sporozoites asvaccines to prevent malaria. Also disclosed are methods of usingpurified pathogenic parasites useful for assessing the effectiveness ofantimalarial drugs and vaccines, and in conjunction with antimalarialagents such as chloroquine, useful for conferring protective immunity.

In an embodiment, methods of using aseptically produced, sterile,infectious, substantially purified, attenuated Plasmodium sporozoites toconfer protective immunity against malaria in human and other mammalianhosts are provided. Such methods and compositions can be used to conferprotective immunity against malaria caused by P. falciparum and otherPlasmodium species, without complications which may result from the useof unpurified preparations.

In an embodiment compositions of substantially purified pathogenicPlasmodium sporozoites and methods of using them as research tools, inclinical testing, and in prophylactic vaccination regimens are provided.

Compositions of live, infectious, attenuated, substantially purifiedsporozoites, as well as dosages, regimens and routes of administrationto human and other mammalian subjects are provided.

In an embodiment purified Plasmodium parasites in excipient areprovided, particularly parasites at the sporozoite stage of development,said parasites metabolically active, infectious, and substantially freefrom contaminant material. In another embodiment, the sporozoites areaseptically prepared. In another embodiment, the sporozoites aresufficiently attenuated to prevent development beyond the liver stage(mature schizont stage) of the parasite life cycle.

In another embodiment, methods are provided for conferring protectiveimmunity in mammalian and human hosts against malaria caused by aPlasmodium species, comprising providing live, metabolically active,infectious, attenuated sporozoites of a Plasmodium species in anexcipient and substantially free of attendant material and administeringat least one dose of said sporozoite preparation to said host; whereinpathological manifestations of malaria are prevented and the host isprotected from developing malaria after subsequent exposure topathogenic Plasmodium sporozoites of said species.

In another embodiment, methods are provided for purifying metabolicallyactive, infectious parasites, particularly Plasmodium sporozoites byproviding an aqueous pre-purification preparation comprising sporozoitesand attendant non-sporozoite material, sequentially passing thepreparation through a set of size exclusion filters comprising: a) afirst exclusion filter with a nominal pore size capable of retainingattendant material larger than 10 microns and allowing passage of thesporozoites; b) a second exclusion filter with a nominal pore sizecapable of retaining attendant material larger than about 0.6 micronsand allowing passage of the sporozoites; and c) a third size exclusionmembrane filter with a precise pore size capable of retainingsubstantially all attendant material larger than 1.2 microns andallowing passage of the sporozoites. Substantially purified sporozoitesare then collected on a collecting filter with a pore size capable ofretaining the sporozoites, and the purified sporozoite preparation isthen eluted from the collecting filter and resuspended at the desiredconcentration.

In another embodiment, compositions useful for determining the efficacyof malaria-related drugs, vaccines and the like are provided. Thecompositions comprise live, substantially purified, pathogenicPlasmodium parasites.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 a—Photo-micrographic observation of crude preparations of PfSPZ(Campaign 3). Photo-micrographs taken at 200× magnification. BeforePurification SGM=837 ng/25,000 PfSPZ (Samples were adjusted to 15×10⁶PfSPZ/ml)

FIG. 1 b—Photo-micrographic observation of purified preparations ofPfSPZ (Campaign 3). Photo-micrographs taken at 200× magnification. AfterPurification SGM=0.23 ng/25,000 PfSPZ (Samples were adjusted to 18×10⁶PfSPZ/ml)

FIG. 2 a—Photo-micrographic observation of crude preparations of PfSPZ(Campaign 6). Photo-micrographs taken at 200× magnification. BeforePurification—SGM=781 ng/25,000 PfSPZ (Samples were adjusted to 17.78×10⁶PfSPZ/ml)

FIG. 2 b—Photo-micrographic observation of purified preparations ofPfSPZ (Campaign 6). Photo-micrographs taken at 200× magnification. AfterPurification—SGM=0.65 ng/25,000 PfSPZ (Samples were adjusted to16.71×10⁶ PfSPZ/ml)

FIG. 3 a—Stage specific expression of PfLSA-1 (P. falciparumsporozoites). PfSPZ incubated with an anti-PfCSP monoclonal antibody.

FIG. 3 b—Stage specific expression of PfLSA-1 (P. falciparumsporozoites). PfSPZ incubated with anti-PfLSA-1 polyclonal antibodies.

FIG. 3 c—Stage specific expression of PfLSA-1 (3 day liver stageparasites). 3 day liver stage parasites incubated with anti-PfLSA-1polyclonal antibodies.

DETAILED DESCRIPTION OF THE INVENTION

There is presently no FDA-approved malaria vaccine. However, resultspublished during the last 30 years have demonstrated that administrationof radiation attenuated P. falciparum sporozoites delivered by the biteof greater than 1,000 P. falciparum-infected mosquitoes provides sterileprotective immunity in greater than 90% of exposed individuals for atleast 42 weeks, and was effective against multiple isolates of P.falciparum from throughout the world. As provocative as theseobservations might have been, they did not suggest a vaccine or anobvious approach to developing a malaria vaccine, because of severaltechnical hurdles, including isolating and purifying sporozoites anddoing so under aseptic conditions. Furthermore, work by a number ofscientists indicated that excellent protection could only be achieved inthe mouse model system by intravenous administration of attenuatedsporozoites. Because parenteral non-intravenous routes of administrationconventionally used in human immunization, e.g. subcutaneous andintramuscular inoculation, did not lead to adequate protective immunityin this mouse model system, it was not considered possible to develop anattenuated sporozoite vaccine for humans. Utilizing a mouse model systemthat is considered more analogous to human malaria caused by P.falciparum, it has been found that parenteral non-intravenousadministration of sporozoites leads to high level protection (USPublication No. 2005/0208078). Many other technical hurdles ofdevelopment for a pharmaceutically acceptable, live attenuated malariavaccine have also been overcome—among them, aseptic production ofsufficient quantities of sporozoites isolated from attendant material(See particularly, Hoffman, S L and T C Luke, U.S. Pat. No. 7,229,627B2, explicitly incorporated herein by reference).

However, a vaccine suitable for routine use in human subjects requires asubstantially pure inoculum and a sporozoite inoculum requiressporozoites that have been substantially purified from the source fromwhich they were produced. The vaccine compositions provided hereincomprise Plasmodium sporozoites in a substantially purified form,substantially free of attendant (source) material, which is not specificto the sporozoites themselves. Additionally, in some embodiments ofvaccine compositions provided herein the vaccine compositions aresubstantially free from contaminating material, microorganisms andexogenous pathogens. Provided are substantially purified sporozoites andmethods of purifying sporozoites, and compositions of purifiedsporozoites and excipient.

DEFINITIONS

The terms “about” or “approximately” means within one standard deviationas per the practice in the art.

“Additive” as used herein as a noun is a compound or composition addedto a sporozoite preparation to facilitate preservation of thepreparation. Additives may include cryoprotectants such as DMSO andglycerol, antioxidants, and the like.

“Aseptic” as used herein means absent the introduction of detectablecontamination of other microorganisms such as bacteria, fungi,pathologic viruses and the like. An aseptic method of sporozoitepreparation results in a sterile preparation of sporozoites—free of anyother type of microorganism or infectious agent. Microbiological assaysused to monitor an aseptic methodology assess the presence or absence ofcontamination. They include, but are not limited to, the MicrobialLimits Test, current USP <61>, incorporated herein by reference.

“Attendant” material as used herein means material in a crudepreparation of parasites which is not specific to the parasites per se.For example in a preparation of sporozoites, attendant material is thatwhich is not specific to the sporozoite per se. Attendant materialincludes material specific to the source from which sporozoites weregrown or produced, particularly biological debris, more particularlyprotein, said material isolated along with the sporozoites in a crudepreparation. That which makes a preparation “crude” is the attendantmaterial in the preparation, and with regard to sporozoites, includesmaterial specific to the substrate from which the sporozoites havedeveloped or from which the sporozoites have been isolated. With regardto sporozoites dissected from and isolated from the salivary glands ofhost mosquitoes, attendant material is the host material, host protein,salivary gland material, saliva and the like. Attendant material doesnot include components which have been intentionally added to apreparation, e.g., excipient, diluent, additives and the like. In someembodiments, a component of attendant material which has been removedfrom a crude preparation may be added back to the purified preparationto optimize sporozoite infectivity sporozoite immunogenicity, orsporozoite presentation to the host. Such added back material is notconsidered attendant as defined herein.

“Attenuate” as used herein means to render a live organism unable tocomplete its life cycle without killing it. The organism may have alimited capacity to replicate, express proteins, and to develop throughsome life cycle stages, but arrests development at a particular lifecycle stage and is unable to developmentally progress beyond that stage.With regard to the attenuated Plasmodium parasites disclosed herein,i.e. parasites which have been exposed to radiation at the sporozoitestage, they retain the ability to infect host hepatocytes and expressstage specific proteins, but are unable to develop beyond liver stage,are unable to reenter the blood stream of infected hosts subsequent toliver-stage (merozoite stage), are unable to cause the disease pathologyof malaria. Other attenuated Plasmodium-species parasites may retain theability to infect host hepatocytes, develop beyond liver stage, reenterthe blood stream of infected hosts subsequent to liver-stage (merozoitestage), and even infect erythrocytes, but subsequently arrestdevelopment prior to the stage at which the disease pathology of malariamanifests itself.

“Conferring protective immunity” as used herein refers to providing to apopulation or a host (i.e., an individual) the ability to generate animmune response to protect against a disease (e.g., malaria) caused by apathogen (e.g., Plasmodium falciparum) such that the clinicalmanifestations, pathology, or symptoms of disease in a host are reducedas compared to a non-treated host, or such that the rate at whichinfection, or clinical manifestations, pathology, or symptoms of diseaseappear within a population are reduced, as compared to a non-treatedpopulation.

“Contaminant” and contamination as used herein means adventitiousmicrobial or viral contamination or pathogenic contamination, includingbut not limited to bacteria, viruses, mycoplasma, fungi and the like. Anaseptic process is designed to prevent the introduction of contaminationand a sterile preparation is absent the presence of contamination.Contaminants may be non-biologic or inorganic toxics as well.Contaminants may be accidentally or inadvertently introduced at any stepduring a process.

“Diluent” as used herein is a medium in which sporozoites and sporozoitecompositions are prepared to achieve a desired concentration. Diluentmay be, for example, normal saline or phosphate buffered saline, ormedia such as medium 199 or medium E199 (medium 199 with Earle'sBalanced Salts).

“Excipient” means an inactive substance used as a carrier, vehicle, ordiluent for the active ingredient of a pharmaceutical preparation suchas a vaccine. As used herein, an excipient is an ingredient added to thepreparation during the isolation and purification process to aid in theprocess, minimize non-specific interactions or adsorptions, and/orprovide volume. Examples of excipients include inactive proteins such asserum albumins, particularly human serum albumin, from whatever source,including but not limited to purified from blood, produced as arecombinant protein or produced synthetically. Sporozoites dissectedfrom mosquito salivary glands may be placed in excipient for furtherprocessing. Excipient may be added to pharmaceutical preparations forseveral reasons including, maintaining activity, increasing stability,or preventing inadvertent loss of isolated sporozoites throughout thepurification process.

“Immune response” as used herein means a response in the recipient tothe introduction of attenuated sporozoites generally characterized by,but not limited to, production of antibodies and/or T cells. Generally,an immune response may be a cellular response such as induction oractivation of CD4+ T cells or CD8+ T cells specific for Plasmodiumspecies epitopes, a humoral response of increased production ofPlasmodium-specific antibodies, or both cellular and humoral responses.With regard to a malaria vaccine, the immune response established by avaccine comprising sporozoites includes but is not limited to responsesto proteins expressed by extracellular sporozoites or other stages ofthe parasite after the parasites have entered host cells, especiallyhepatocytes and mononuclear cells such as dendritic cells and/or tocomponents of said parasites. In the instant invention, upon subsequentchallenge by infectious organisms, the immune response preventsdevelopment of pathogenic parasites to the asexual erythrocytic stagethat causes disease.

“Intravenous” as defined herein means intentional introduction, directlyinto the lumen of an identified large blood vessel such as a vein.

“Metabolically active” as used herein means alive, and capable ofperforming sustentative functions and some life-cycle processes. Withregard to attenuated sporozoites this includes but is not limited tosporozoites capable of invading hepatocytes in culture and in vivo,potentially having a limited capacity to divide and progress throughsome developmental stages, and de novo expressing stage-specificproteins.

“Mitigate” as defined herein means to substantially reduce, or moderatein intensity, symptoms and/or pathology of malaria which might otherwisemanifest subsequent to vaccination.

“Nominal” as defined herein and with regard to the pore size of a filtermeans the effective pore size and refers to the apparent pore size forpassage of globular particles through the filter such that 90% of theparticles of said size are excluded by the filter.

“Parenteral” as defined herein means not through the alimentary canal,but rather by introduction through some other route, as intradermal,subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal,intrasternal, intravenous, transcutaneous and the like.

“Prevent” as defined herein and used in the context of preventingmalaria means to keep a majority, up to all, of the pathology andclinical manifestations of malaria from manifesting.

“Purify” as defined herein and with regard to preparations ofsporozoites, means to separate from attendant material or to separatefrom material considered to be undesirable.

“Sterile” as defined herein and with regard to sporozoites, means absentany contaminating microorganism. A sterile preparation of sporozoites isobtained by means of an aseptic preparation methodology. The sterilityof a preparation is determined by specific assays, including but notlimited to the Sterility Test current USP <71>, incorporated herein byreference.

“Substantially purified” with regard to a preparation of sporozoitesmeans reduction in the amount of attendant contamination to less than 85ng/25,000 sporozoites or at least an 18 fold reduction in attendantcontamination. Satisfaction of either of the aforementioned criteria issufficient to render a preparation of sporozoites “substantiallypurified.”

“Therapeutic” as defined herein relates to reduction of clinicalmanifestations or pathology which have already become manifest. A“therapeutically effective amount” as used herein means an amountsufficient to reduce the clinical manifestations, pathology, or symptomsof disease in an individual, or an amount sufficient to decrease therate at which clinical manifestations, pathology, or symptoms of diseaseappear within a population.

“Vaccine” as used herein is a preparation comprising an immunogenicagent and a pharmaceutically acceptable diluent potentially incombination with excipient, adjuvant and/or additive or protectant. Theimmunogen may be comprised of a whole infectious agent or a molecularsubset of the infectious agent (produced by the infectious agent,synthetically or recombinantly). When the vaccine is administered to asubject, the immunogen stimulates an immune response that will, uponsubsequent challenge with infectious agent, protect the subject fromillness or mitigate the pathology, symptoms or clinical manifestationscaused by that agent. A therapeutic (treatment) vaccine is given afterinfection and is intended to reduce or arrest disease progression. Apreventive (prophylactic) vaccine is intended to prevent initialinfection or reduce the rate or burden of the infection. Agents used invaccines against a parasitic disease such as malaria may be whole-killed(inactive) parasites, live-attenuated parasites (unable to fullyprogress through their life cycle), or purified or artificiallymanufactured molecules associated with the parasite—e.g. recombinantproteins, synthetic peptides, DNA plasmids, and recombinant viruses orbacteria expressing Plasmodium proteins. A vaccine may comprisesporozoites along with other components such as excipient, diluent,carrier, preservative, adjuvant or other immune enhancer, orcombinations thereof, as would be readily understood by those in theart.

Purification of Sporozoites

Plasmodium-species parasites are grown aseptically in cultures as wellas in vivo in Anopheles-species mosquito hosts, most typically Anophelesstephensi hosts. Methods of axenically culturing Plasmodium-speciesliver stage parasites (Kappe et al. US Pub. 2005/0233435) and methods ofproducing attenuated and non-attenuated Plasmodium-species sporozoites,particularly, methods of growing and attenuating parasites inmosquitoes, and harvesting attenuated and non-attenuated sporozoites areknown in the art and have been described (See, Hoffman & Luke, U.S. Pat.No. 7,229,627; US Pub. No. 2005/0220822.

The purification aspect of the invention separates sporozoites fromattendant material such as mosquito host salivary gland material(hereinafter, “SGM”), mosquito-specific material, and any othernon-sporozoite material or component, thereby achieving a substantiallypurified preparation containing sporozoites. As an aspect ofpurification, sporozoites are concentrated relative to attendantcontaminating material such as SGM. Here, the purity of a preparation ismeasured using an enzyme-linked immunosorbent assay (ELISA) to quantifyattendant antigenic SGM present in crude and purified preparations ofsporozoites (hereinafter, “SGM-ELISA”). Other methods, such as, but notlimited to, capillary electrophoresis, mass spectrometry, reverse phasechromatography, immunoblotting, light-scattering and UV spectrometry,may be similarly adapted to measure attendant material. With regard tothe purification of sporozoites from sources other than mosquitoes,similar assays, such as ELISA, relevant to the attendant material in thecrude preparation from which sporozoites are to be purified, would besimilarly adapted. The development of such ELISA assays adapted toantigens of attendant material from sources other than mosquito salivaryglands would use a similar protocol, commonly known to those in the art.To illustrate, a protocol for an impurity assay of attendant SGM isprovided. It is to be understood that this protocol is provided forillustration and may be modified e.g. by scaling up, or otherwisemodified, and is not intended to limit the scope of the invention asclaimed.

Purification Methods

Disclosed herein are methods of purifying live Plasmodium species, e.g.falciparum, vivax, malariae, or ovale.

The methods disclosed utilize a series of size exclusion filters ofdifferent types and with different pore sizes, assembled in a novel andnon-obvious fashion. The methodology eliminates attendant material frompreparations of live, motile parasites. An aspect of this method is thatthe pore size of a size exclusion filter in sequence is not alwayssmaller than the pore size of the size exclusion filter which precedesit. Another aspect is that some filters provide a matrix with a nominalpore size and at least one filter provides a track-etched filter with aprecise pore diameter. At least one filter has a pore size close to orslightly smaller than the diameter of the parasite.

By way of example, the purification of Plasmodium-species sporozoites isdisclosed; however, it will be understood by those practiced in the artthat, based upon the physical size of the organism, adjustment of thefilter pore size will yield a similar purification. For example, thePlasmodium falciparum sporozoite is rod-shaped, and about 0.8±0.2 μm indiameter and 8.5±1.5 μm in length. Hoffman, S L et al In TropicalInfectious Disease 2nd Ed. 2006, Elsevier, Philadelphia, Pa. pg 1027;Xu, L. H. et al 1985 Zoo. Res. 6:33-6.

Typically in a preparation for purification, the salivary glands from150 to 400 mosquitoes are dissected. The sporozoites are released fromthe salivary glands by passage back and forth in a needle and syringe(trituration), and sporozoites from these glands are collectivelypurified. However, several fold more mosquitoes may be dissected inscaled up preparations, in an embodiment up to 1,000 mosquitoes, inanother embodiment up to 5,000 mosquitoes, in another embodiment up to10,000 mosquitoes. Sporozoites are released from salivary glands bytrituration and the triturated salivary gland preparations(pre-purification preparations) are purified by the size exclusionfiltration process disclosed herein. Sporozoites are maintainedthroughout the purification process in an excipient, typically onepercent human serum albumin (HSA) in Medium 199 with Earle's salts(E-199).

A—Preparation of Material for Purification

The triturated dissection product (pre-purification preparation) isreceived altogether in a single tube at a time. This is the SGMpre-purification preparation. It represents about 100,000 to 1 billionsporozoites, preferably at least 1 million sporozoites and morepreferably at least 25 million sporozoites. The measured amount of SGMin the pre-purification preparation is usually between 300 ng and 12,000ng per 25,000 sporozoites, more typically, between 400 ng and 1,100 ngper 25,000 sporozoites. The pre-purification preparation is then dilutedto 10 ml with excipient. Solutions and samples are kept between 15-30°C. for the duration of the purification.

B—Purification Procedure

Using a peristaltic pump the diluted pre-purification preparation ispumped across a series of size exclusion filters at a flow rate of atleast 1 ml/min but no more than 1000 ml/min, preferably at least 2ml/min, but no more than 500 ml/min, and more preferably with a flowrate of at least 3 mL/min and no more than 200 mL/min. The correspondingflux across each filter is at least 1 L/hr/m2 but no more than 2000L/hr/m2, preferably 3 L/hr/m2 to 1500 L/hr/m2, and most preferably atleast 125 L/hr/m2 but no more than 250 L/hr/m2. Filters are connected inseries, usually with medical grade silicone tubing. Preferably, theinitial filter (Filter #1) or the initial two filters (Filters #1 and#2) are matrix filters and are made of polypropylene, however, nylon,mixed cellulose ester and borosilicate glass or other material known tothose in the art may be used. Preferably, the penultimate filter(Filter#3) is a membrane filter, most preferably a track-etchedpolycarbonate filter, although other filters with similar propertiesknown to those in the art may be used. For aseptic procedures, thefilters are sterile. In an embodiment, three filters (Filter #1, Filter#2 and Filter #3) are connected in series and sporozoites are capturedby dead end filtration on Filter #4. Additional filters may be used.Alternatively, only one or two filters may be used (as discussed belowin Paragraph 59), however using three filters is optimal. In anembodiment, filter #1 is a membrane matrix with a nominal pore size ofat least about 2.5 microns, but no more than about 30 microns,preferably at least about 5 microns, but no more than 20 microns. In oneembodiment the filter used has a nominal pore size of about 10 micronswith a filtration area of 17.5 cm². (Polygard®-CN Optiscale—MilliporeCat. No. SN1HA47HH3). In a scaled up embodiment the filtration area is1800 cm2. The nominal pore size of Filter #2 (also a membrane matrix) isat least about 0.3 microns but not larger than about 1.2 microns. In oneembodiment, the pore size is about 0.6 micron with a filtration area of17.5 cm2. (Polygard®-CN Optiscale filter—Millipore Cat. No.SN06A47HH3)—smaller than the diameter of Plasmodium. In a scaled upembodiment the filtration area is 1800 cm2. In one embodiment, Filter #3is a track-etched membrane filter with precise pore diameter andconsistent pore size, and has a pore size of at least 1.2 microns butnot larger than 3 microns—larger than the nominal pore size of thepreceding filter. In one embodiment the filter used has a pore size of1.2 microns with a filtration area of 11.3 cm2. (Isopore membrane, 47 mmin diameter—Millipore Cat. No. RTTP04700) held in a Swin-Loc filterholder (Whatman Cat. No. 420400). In a scaled up embodiment thefiltration area is 127 cm2. Filtered material is captured on Filter #4in a stirred ultrafiltration cell (Millipore, model 8200) fitted with anIsopore membrane, 90 mm in diameter with a filtration area of 28.7 cm2,and a track-etched pore size of no more than 0.8 microns, preferably nomore than 0.6 microns, and preferably no more than 0.2 microns. In oneembodiment the pore size is 0.4 microns (Millipore Cat. No. HTTP09030).In a scaled up embodiment the filtration area is 162 cm2. In anotherscaled up embodiment the filtration area is 63 cm2. The system is washedseveral times with media. When the retentate volume reaches about 40 mlin the stirred cell, the stirred cell container outlet is opened anddrained by gravity leaving about 5-10 ml of residual retentate althoughthe retentate volume can be reduced by other methods such as applyingpressure from compressed gas such as nitrogen or a mechanical devicesuch as a piston, gravity is the preferred method. This residualretentate is collected and transferred, together with three washes usingpurification media to a total of about 35 ml, typically in a sterile 35ml Oak Ridge or similar centrifuge tube (the size of the tube will varydepending on the volume of the preparation). Purified sporozoites inmedia in the 35 ml Oak Ridge tube are centrifuged at 5,000 g to 25,000g, preferably at 16,300 g, for 2 minutes to 12 minutes, preferably fiveminutes, to pellet the sporozoites. The supernatant media is decanted.This step additionally purifies the sporozoite preparation by removingsmaller more buoyant materials and soluble materials that remain in thesupernatant.

Using the 3 size-exclusion filter methodology, this procedure providesgreater than a substantial reduction of attendant material in thepurified sporozoite preparation relative to the attendant material inthe pre-purification preparation (reduction factor) of from 200 to10,000 fold. The amount of residual SGM in purified preparations ofsterile purified sporozoites routinely is less than 25 ng of attendantmaterial per 25,000 sporozoites (greater than 97% reduction relative tothe initial amount of SGM), preferably less than 15 ng per 25,000 (98%reduction) sporozoites, and more preferably less than 1 ng per 25,000sporozoites (99.9%). The contaminating SGM in each purified preparationdescribed herein is usually reduced several thousand fold relative toSGM in the initial triturated pre-purified salivary gland material fromwhich each purified preparation is derived. Preferably the purificationreduction factor is at least 15-fold, more preferably, the purificationfactor is at least 1,500-fold and most preferably at least 3,500-fold.The geometric mean of the reduction factor in the 10 campaigns describedin Example 1 is 1625, a 99.93% reduction in SGM during the purificationprocess.

For example, purified sporozoite preparations, isolated from dissectedmosquito salivary glands shown in Example 1 (Table 2) contained a rangeof from 0.19 ng to 1.01 ng SGM per 25,000 sporozoites and the percentagereduction of attendant SGM ranged from 99.91% to 99.97%. Alternatively,the method described in Paragraph 59 below provided a preparation ofsporozoites with no more than 85 ng SGM per 25,000 (95% reduction ofattendant SGM material) and are considered substantially purified. Thegeometric mean of SGM in purified preparations from the 10 productioncampaigns in Table 2 (using 3 size exclusion filers) was 0.51 ng SGM per25,000 sporozoites with a 95% confidence interval of from 0.34 to 0.67ng SGM per 25,000 sporozoites.

In an alternative embodiment, Filter #3 is eliminated from the filtertrain described in Paragraph 56 above and a 2 size-exclusion filtermethodology (Intermediate Filter #1 and Filter #2) is used. Sporozoitesare captured as described in Paragraph 56 above. In an example of thisembodiment, a crude preparation of 1,555 ng/25,000 sporozoites wasloaded and purified as described (minus Filter #3). After collection(either on a 0.4 micron or 0.45 micron filter) and resuspension, thepurified preparation contained 84 ng/25,000 sporozoites. Here, thereduction factor was greater than 18 fold and 94.6% of SGM contaminationwas removed. This preparation is considered substantially purified.

The range of the yield of purified sporozoites was between 30% and 100%of the number of sporozoites in the starting preparation—usually between50% and 70%.

This method is effective at reducing contamination in a crude sporozoitepreparation. The sporozoites are then preserved. In an embodiment, thesporozoites are cryopreserved (Leef J. L. et al. Bull WHO 57 (suppl 1)(1979) 87-91; Orjih A. U., F. and Nussenzweig RS. Am. J. TroP. Med. Hyg.(1980) 29(3): 343-7). In an embodiment, the sporozoites are preserved bylyophilization. In an embodiment, the sporozoites are preserved byrefrigeration. Other methods of preservation are known to those skilledin the art.

Attenuation

Several means of inducing Plasmodium sporozoite attenuation are known.These include heritable genetic alteration, gene mutation, radiationexposure, and exposure to mutagenic chemicals, exposure to metabolicinhibition chemicals and exposure to environmental conditions such ashigh or low temperature or pressure.

Methods of inducing attenuation of sporozoites by radiation exposurehave been disclosed (See, e.g., Hoffman & Luke, U.S. Pat. No. 7,229,627;US Pub. No. 2005/0220822) and are incorporated herein by reference.Sporozoites may be attenuated by at least 100 Gy but no more than 1000Gy, preferably 120 to 200 Gy, and most preferably about 150 Gy. Theattenuation of Plasmodium parasites of the vaccine disclosed hereinallow sporozoite-stage parasites to remain metabolically active,infectious with the ability to invade hepatocytes (potency); whileensuring that parasites do not develop to the fully mature liverschizont stage, cannot reenter the host bloodstream, invade erythrocytesor reach the developmental stages which cause disease (safety). Those ofskill in the art can routinely determine the developmental stages of theparasite and adjust the attenuation as necessary.

The biology of the Plasmodium infection of hepatocytes providesopportunities for in vitro assays to demonstrate both potency andsafety. Normally, sporozoites migrate from the bite site of the mosquitoto the liver, primarily via the blood stream, but potentially via thelymphatic system. In the liver they multiply within hepatocytes,producing, in the case of P. falciparum, about 10,000-40,000 progeny(merozoites) per infected cell. These liver stage parasites expressmultiple proteins, which are not expressed in sporozoites or at laterdevelopmental stages. After developing in the liver to the merozoitestage, they are released from hepatocytes and reenter the blood stream,expressing a different set of stage-specific proteins—different fromthose expressed during the sporozoite and early hepatic stages—andinvade erythrocytes, where additional multiplication increases parasitenumbers by approximately 10 to 20 fold every 48 hours. Unlike the fiveto ten day development in the liver, which does not induce any symptomsor signs of illness, untreated blood stage infection causes hemolysis,shaking chills, high fevers, and prostration, and many other symptomsand signs of malaria.

Potency Assay

The level of infectious potency of purified radiation-attenuated P.falciparum sporozoites may be assessed by measuring the expression of P.falciparum Liver Stage Antigen-1 (PfLSA-1) in human hepatocyte culturesusing an immunofluorescence assay (IFA). PfLSA-1 is a protein that isnot expressed by sporozoites, but is expressed by P. falciparumparasites (both attenuated and wild type) after they have successfullyinvaded hepatocytes (FIG. 3). Expression of PfLSA-1 indicates that theattenuated sporozoites are metabolically active.

Human hepatocytes (cell-line HC-04 [1F9], See Prachumsri, J. and N.Yimamnuaychok, U.S. Pat. No. 7,015,036) are seeded approximately 24±6hours pre-infection using a 1:1 ratio of Dulbecco's modified Eagle'smedium and F-12 Ham's mixture supplemented with 10% fetal bovine serumand 2% penicillin/streptomycin solution (HC-04 growth medium). The cellsare seeded at a concentration of 4.0×104 cells/0.3 ml/well into 8-wellLab Tek Permanox (NUNC) chamber slides coated with Entactin Collagen-IVLaminin (ECL) (Upstate) and incubated at 37±1° C., 5% CO2 and 80%relative humidity. When the monolayer confluency reaches ≧80% at 24±6hours post-seeding, the culture supernatant is aspirated from each well,replaced with 0.3 ml of fresh HC-04 growth medium and returned to theincubator.

Attenuated P. falciparum sporozoites are diluted with HC-04 growthmedium to a concentration of 500 sporozoites/μl. Fifty μl of the dilutedparasite sample is then added to each well, after aspirating 300 μl ofmedium, allowing a total of 2.5×104 cells to infect the HC-04 cells ineach well. Infected hepatocytes are incubated for 3±0.5 hours, washedthree times with HC-04 growth medium by gentle aspiration of thesupernatant and cultured with 0.3 ml of fresh medium per well. Culturesare observed daily for three days for any indication of the presence ofcontamination. In the absence of contamination, the culture supernatantis aspirated from individual wells and replaced with 0.3 ml of freshHC-04 growth medium daily and returned to the incubator. At 72±6 hourspost infection, cultures are observed microscopically for possiblecontamination. In the absence of contamination, the cultures are washedthree times using 0.3 ml of phosphate buffered saline (PBS), fixed atroom temperature with 0.3 ml of cold methanol (stored at −20° C.),washed another three times using 0.3 ml of PBS and stored in 0.3 ml ofthe same. Slides can be stored in a refrigerator at 2-8° C. for up to 72hours until they are stained for immunofluorescence using ananti-PfLSA-1 polyclonal rabbit serum as the primary antibody and anAlexa fluor-488 labeled anti-rabbit secondary antibody. The plasticchamber and gasket are removed from stained slides. Slides are mountedusing Vectashield mounting medium (Vector) and covered with a coversliP. Slides are evaluated by counting all parasites expressing PfLSA-1per well using an epifluorescence microscope with phase contrast (FIG.3). The sporozoites are considered potent if there is no contaminationof the preparations at any step and the number of fluorescing parasitesis at least 200/well.

Vaccine Compositions

Pharmaceutical compositions comprising live Plasmodium sporozoites, bothattenuated and pathogenic, and methods of using these compositions aspreventive vaccine compositions to prevent disease, and as pathogenicchallenge compositions to infect volunteers in the testing of vaccinesand drugs, have been provided (see particularly Hoffman USSNUS2005/0220822). Various categories of attenuated sporozoites have beenconsidered for use in vaccines. These include sporozoites attenuated byvarious methods including heritable genetic alteration, gene mutation,radiation exposure, and chemical exposure. Various attenuated isolatescreated by direct genetic manipulation of the parasites have beendescribed for P. falciparum (van Schaijk et al. PLoS ONE (2008) 3:e3549)as well as murine-specific Plasmodium species (Kappe et al. U.S. Pat.No. 7,122,179; van Dijk et al. PNAS (2005) 102:12194-12199; Labaied etal. Infect Immun. (2007) 75:3758). In an embodiment, radiationattenuation of human-specific Plasmodium is achieved by exposure togamma radiation. (Hoffman, S. L. et al. (2002) 185:1155-1164). Thepurification processes provided herein are intended for purification offully infectious sporozoites as well as attenuated sporozoites.

In an embodiment, attenuated Plasmodium sporozoites may be geneticallymanipulated to contain exogenous genes of other Plasmodium-species or ofother pathogenic organisms which may be expressed prior to, during orsubsequent to infection.

Vaccination methodologies comprising purified pathogenic sporozoites arealso contemplated. For instance, pathogenic sporozoites may beadministered to individuals concurrently treated with anti-malarials(e.g. chloroquine) effective against the asexual erythrocyte stageparasites, or administered to individuals subsequently treated withanti-malarials effective against the asexual erythrocyte stageparasites, thereby preventing the pathology caused by the parasites invivo, while allowing the parasites to stimulate protective immuneresponses. Roestenberg, M., et al. (2009) NEJM 361:468-478; Pombo, D. J.et al. (2002) Lancet 360:610-617.

Sterile, purified pathogenic sporozoites are also useful in challengeprotocols to assess the effectiveness of vaccines and vaccinationmethodologies in challenge studies of volunteers previously treated withthese vaccines and vaccine methodologies. Similarly, purified pathogenicsporozoites are useful to generate the symptoms, signs or pathology ofmalaria in studies to assess the effectiveness of chemoprophylacticdrugs and therapeutics.

Sterile live pathogenic sporozoites that have been purified, as well assterile live attenuated sporozoites that have been purified aregenerally more useful than their non-sterile, unpurified counterpartsbecause compositions and vaccines comprising sterile purifiedsporozoites reduce or eliminate the risk that attendant material orcontamination will cause unwanted immune responses, adventitiousinfections or other unexpected consequences. Vaccines comprisingpurified live attenuated Plasmodium species sporozoites usually areadministered parenterally, and by other routes as described herein. Suchvaccines are useful for prevention or reduction of severity of malaria,its manifestations, symptoms or its pathology.

In an embodiment, compositions and vaccines comprising asepticallyprepared attenuated purified sporozoites provide partial, enhanced, orfull protection in human and other mammalian subjects not previouslyexposed to a malaria-causing pathogen, or exposed, but not fullyprotected. These compositions and vaccines are similarly useful toreduce the chance of developing a disease-producing infection fromparasites that causes malaria, including species of Plasmodium, e.g. P.falciparum or P. vivax, and the like, and reduce the chance of becomingill when one is infected, reduce the severity of the illness, such asfever, when one becomes infected, reduce the concentration of parasitesin the infected person, or reduce mortality rates from malaria inpopulations exposed to malaria parasites. In many cases even partialprotection or delay in the time it takes an immunized individual ascompared to a non-immunized individual to become infected with theparasites or ill from infection is beneficial. Similarly, a vaccinetreatment strategy that results in any of these benefits in about 30% ofa population may have a significant impact on the health of a communityand of the individuals residing in the community. In other embodimentscomprising pathogenic sporozoites, compositions disclosed are useful fordemonstrating the efficacy of vaccines, drugs for the treatment ofmalaria, and other treatments and preventions of malaria.

Provided are methods for prevention of malaria in a subject. The methodscomprise administering to the subject a vaccine which has been preparedaseptically and comprises substantially purified live attenuatedPlasmodium sporozoites in an amount effective to prevent malaria.

The subject to which the vaccine is administered in accordance withthese methods may be any human or other mammal, susceptible to infectionwith a malaria parasite. For such methods, administration can be via thealimentary tract, such as oral, or administration can be parenteral,including, but not limited to mucosal, intranasal, epidermal, cutaneous,intramuscular, subcutaneous, intradermal, submucosal, intravenous andthe like. Moreover, the administration may be by continuous infusion orby single or multiple boluses as well as delivery mediated bymicroneedles.

The prevention and/or treatment of malaria may be readily ascertained bythe skilled practitioner by evaluation of clinical or pathologicalmanifestations associated with malarial infection, for example elevatedtemperature, headache, fatigue, coma, or percent of erythrocytesparasitized. Thus, according to the methods of the present invention,the subject shows improved or absent clinical signs, symptoms orpathological manifestations of malaria following administration of avaccine comprising purified live attenuated Plasmodium sporozoites.

Effective and optimal dosage ranges for vaccines and immunogens can bedetermined using methods known in the art. Guidance as to appropriatedosages to achieve an anti-malarial effect is provided from theexemplified assays disclosed herein. More specifically, results from theimmunization pattern described herein and in cited references can beextrapolated by persons having skill in the requisite art to provide atest vaccination schedule. Volunteer subjects are inoculated withvarying dosages at scheduled intervals and test blood samples areevaluated for levels of protection against malaria upon subsequentchallenge with infective parasites. Such results can be used to refinean optimized immunization dose and dosage regimen (schedule) foreffective immunization of mammalian, specifically human, subjects. Adose effective in conferring a protective immunity is from 5,000 to400,000 purified attenuated sporozoites administered in a dosage regimenof 1 to 6 doses, more particularly a dose of from 15,000 to 270,000purified attenuated sporozoites in a dosage regimen of at least 2 doses,and most particularly a dose of from 25,000 to 150,000 purifiedattenuated sporozoites in a dosage regimen of 3 or 4 doses.

An immune response in a subject can be measured by standard testsincluding, but not limited to the assessment of humoral and cellularimmune responses, including, but not limited to: measurement of antigenspecific or parasite stage specific antibody responses; directmeasurement of peripheral blood lymphocytes by means known to the art;natural killer cell cytotoxicity assays (Provinciali et al. (1992) J.Immunol. Meth. 155: 19-24), cell proliferation assays (Vollenweider etal. (1992) J. Immunol. Meth. 149: 133-135), immunoassays of immune cellsand subsets (Loeffler et al. (1992) Cytom. 13: 169-174; Rivoltini et al.(1992) Can. Immunol. Immunother. 34: 241-251); and skin tests for cellmediated immunity (Chang et al. (1993) Cancer Res. 53: 1043-1050).Various methods and analyses for measuring the strength of the immunesystem have been described, for example, Coligan et al. (Ed.) (2000)Current Protocols in Immunology, Vol. 1, Wiley & Sons.

The vaccines provided comprise aseptic and non-aseptic compositions ofpurified live attenuated Plasmodium sporozoite substantially free ofattendant material, and compositions with a pharmaceutically acceptablediluent, excipient, or carrier. These vaccines are effective inpreventing or mitigating malaria upon subsequent challenge withinfectious parasites. Methods of formulating pharmaceutical compositionsand vaccines are well known to those of ordinary skill in the art (see,e.g., Remington, The Science and Practice of Pharmacy 21st Edition,Hendrickson, ed. (USIP: 2005)).

Comprehended by the invention are vaccine compositions, asepticallyprepared or otherwise, comprising purified, live attenuated ornon-attenuated Plasmodium sporozoites along with appropriate diluent andbuffer. Diluents, commonly Phosphate Buffered Saline (PBS), or NormalSaline (NS), are of various buffer content pH and ionic strength. Suchcompositions may also include an excipient such as serum albumin,particularly human serum albumin. Serum albumin may be purified fromnaturally occurring sources such as human blood, or be produced byrecombinant DNA or synthesis technologies. Such compositions may alsoinclude additives such as anti-oxidants e.g., ascorbic acid, sodiummetabisulfite, and/or preservatives or cryopreservatives. Incorporationof the material into particulate preparations of polymeric compoundssuch as polylactic acid, polyglycolic acid, etc. or into liposomes mayalso be used. (See, e.g., Remington's Pharmaceutical Sciences, 18th Ed.(1990), Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712 whichare herein incorporated by reference.

In order to determine the effective amount of the vaccines, the ordinaryskilled practitioner, considering the therapeutic context, age, andgeneral health of the recipient, will be able to ascertain properdosing. The selected dosage depends upon the desired therapeutic effect,on the route of administration, and on the duration of the treatmentdesired. Experiments to determine levels for dosages can be ascertainedby one of ordinary skill in the art by appropriate human clinical trialsin which various dosage regimens are evaluated for their capacity toelicit protection against malaria.

Disclosed vaccines and disclosed methods of using these vaccines may beuseful as one component in a vaccine regimen, each component in turncomprising a discrete vaccine to be administered separately to asubject. Regimens may include sequential immunization with attenuatedPlasmodium species sporozoites and other types of Plasmodium vaccines,so-called, prime-boost strategies. This may include attenuatedsporozoites as a prime, and Plasmodium-related recombinant protein orproteins in adjuvant as a boost or vice versa. This may also includePlasmodium-related DNA vaccines or a recombinant virus, such asadenovirus, that express Plasmodium-related proteins, as a prime andpurified, attenuated sporozoites vaccine as a boost, or vice versa. Itmay also include sequential or mixed immunization with attenuatedPlasmodium species sporozoites and some form of erythrocytic stageparasites, including, killed and live attenuated. A vaccine complexcomprising separate components may be referred to as a vaccine regimen,a prime/boost regimen, component vaccine, a component vaccine kit or acomponent vaccine package, comprising separate vaccine components. Forexample, a vaccine complex may comprise as a component, a vaccinecomprising purified, aseptic, live attenuated sporozoites. The complexmay additionally comprise one or more recombinant or synthetic subunitvaccine components, including but not limited to recombinant protein,synthetic polypeptide, DNA encoding these elements per se orfunctionally incorporated in recombinant virus, recombinant bacteria, orrecombinant parasite. A vaccine component may also include asepticattenuated axenic sporozoites that are allowed to develop to the earlyliver stage extracellularly.

P. falciparum strains from different parts of the world—West Africa,East Africa, SE Asia, and the like, have been described. Volunteersimmunized with one strain of attenuated sporozoite exhibit protectionagainst others strains (Hoffman, S. L. et al (2002) J. Inf. Dis.185:1155-1164). In an embodiment, multiple isolates and/or strains of aPlasmodium species are combined in a sporozoite composition or in avaccine formulation.

Several Plasmodium species are known to cause malaria in humans,predominantly P. falciparum and P. vivax. Other Plasmodium species causemalaria as well, including P. malariae, and P. ovale. P. knowlesi isalso known to cause human disease. In an embodiment, two or morePlasmodium species are combined in a vaccine formulation. In still otherembodiments, separate components of a vaccine regimen may be derivedfrom different species, e.g., some doses from P. falciparum and othersfrom P. vivax.

In another embodiment, the sporozoite may be a transgenic or recombinantparasite, i.e. a parasite that includes and expresses a DNA sequence orgene that is foreign to the species of parasite which contains it,referred to herein as a transgene. (See, e.g. Franke-Fayard, et al.Molec. and Biochem. Parasitol. 2004 A Plasmodium berghei reference linethat constitutively expresses GFP at a high level throughout thecomplete life cycle. 137:23-33; Wengelnik, K. et al. The EMBO J. 1999The A-domain and the thrombospondin-related motif of Plasmodiumfalciparum TRAP are implicated in the invasion process of mosquitosalivary glands. 18:5195-5204). It will be understood by those skilledin the art that a transgenic parasite, i.e. containing a transgene, canbe purified according to the methodology disclosed herein in the samefashion and to the same degree as the parental parasite from which thetransgenic parasite was created.

Recently published results report cross-species protection in a rodentmalaria system that has been shown to have a high degree ofpredictability for what occurs with the human malaria system (Sedegah,M. et al (2007) Parasite Immunol. 29:559-565). This suggests that avaccine composition comprising P. falciparum may provide protectiveimmunity against P. vivax and/or other human Plasmodium species.

Pharmaceutical compositions may be preserved, cryopreserved,lyophilized, spray-dried, refrigerated, or thermo-stabilized at roomtemperature and the like.

Both the foregoing description and the following examples are exemplaryand explanatory only and are not restrictive of the invention, asclaimed. Moreover, the invention is not limited to the particularembodiments described, as such may, of course, vary. Further, theterminology used to describe particular embodiments is not intended tobe limiting, since the scope of the present invention will be limitedonly by its claims.

Unless defined otherwise, the meanings of all technical and scientificterms used herein are those commonly understood by one of ordinary skillin the art to which this invention belongs. One of ordinary skill in theart will also appreciate that any methods and materials similar orequivalent to those described herein can also be used to practice ortest the invention. Further, all patents, patent applications andpublications mentioned herein are incorporated herein by reference.

As used herein and in the appended claims, the singular forms “a,” “or,”and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “an attenuated sporozoitevaccine” includes a plurality of such sporozoites and reference to “theagent” includes reference to one or more agents and equivalents thereofknown to those skilled in the art, and so forth.

Furthermore, sporozoites which are metabolically active, and alive butattenuated in their life cycle and ability to cause the clinicalmanifestations and pathology of malaria are variously referred to asattenuated, live attenuated and metabolically active, live attenuated.

The numerical parameters set forth in the specification and claims areapproximations that may vary depending upon the desired properties ofthe present invention. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should at least be construed in lightof the number of reported significant digits, applying ordinary roundingtechniques. Nonetheless, the numerical values set forth in the specificexamples are reported as precisely as possible. Any numerical valuearrived at from an experimental measurement, however, inherentlycontains certain errors from the standard deviation of its experimentalmeasurement.

Many modifications and variations of the present invention are possiblein light of the above teachings. It is therefore to be understood that,within the scope of the appended claims, the invention many be practicedotherwise than as specifically described.

The following examples further illustrate the invention. They are merelyillustrative of the invention and disclose various beneficial propertiesof certain embodiments of the invention. These examples should not beconstrued as limiting the invention.

EXAMPLES Example 1 Attendant SGM Determination—ELISA

Sporozoites of any Plasmodium species may be purified by the methodsprovided. The examples provided herein describe the purification of thesporozoites of P. falciparum (PfSPZ). However, other embodiments utilizeP. vivax, P. ovale, P. malariae, and/or P. knowlesi. Still otherembodiments utilize mixtures of these parasites. Still other embodimentsutilize attenuated sporozoites of each species. Still other embodimentsutilize attenuated sporozoites that include and express a DNA sequenceor gene that is foreign to that parasite.

A—Preparation of Anti-SGM Rabbit Anti-SERA

i) Extraction of Salivary Glands from Mosquitoes

To generate rabbit anti-sera against mosquito salivary glands, 10 to 14day old insectary-raised Anopheles stephensi mosquitoes were immobilizedby placing in a 4 C refrigerator for approximately five minutes.Immobilized mosquitoes were placed briefly in a Petri dish containing70% ethanol and then transferred into a Petri dish containing 1×phosphate buffered saline (PBS). A pair of salivary glands from eachmosquito was dissected by hand in 1×PBS on a microscope glass slide andtransferred to a 1.7 ml clear sterile microtube (Axygen Scientific Inc.CA) containing 20-30 μl of 1×PBS. On a daily basis, approximately 100mosquitoes were dissected and 100 pairs of salivary glands in 30 μl PBSwere placed in a freezer at −70 C. When enough salivary glands had beenobtained for one immunization, vials were removed from the freezer,thawed to room temperature and pooled.

ii) Salivary Gland Processing

For the primary inoculation, salivary glands were dissected from 1,000mosquitoes and stored at −70° C. On the day of inoculation, samples werethawed, pooled and lysed by three additional freeze-thaw cycles using adry ice/ethanol bath and thawing at room temperature. The salivaryglands were reconstituted in a final volume of 600 μl of 1×PBS andemulsified with Montanide ISA 720 (SEPPIC, Inc., Fairfield, N.J.).

For all subsequent immunizations, salivary glands were dissected from500 mosquitoes and stored at −70° C. daily. On the day of inoculation,samples were thawed, pooled and homogenized with a pestle (KontesEF2488A). The pestle was rinsed with 100 μl of PBS which was added tothe pool. The sample was reconstituted to a final volume of 600 μl PBSand emulsified within 30 minutes after reconstitution.

iii) Salivary Gland Emulsification

Two ml glass vials (two vials for adjuvant control; two vials forsalivary glands in adjuvant) were obtained. Seven hundred fifty μl ofMontanide ISA 720 adjuvant was drawn through a 5 micron filter syringe.Seven hundred μl of the filtered adjuvant was injected into each glassvial. Three hundred μl of PBS was added to the control vial and threehundred μl of salivary glands in PBS to the experimental vial. The vialswere covered with a rubber stopper. Each vial was further capped with analuminum seal cap and the seal was pressed with a crimper. Vials wereplaced in the small holes of a vortex pad, taped down, and vortexed for30 minutes.

iv) Emulsion and Inoculation Quantities

For the primary immunization two glass vials were used to emulsifyantigen from 1,000 mosquitoes, (each containing 1,000 μl of a 300:700volume ratio of salivary gland/PBS: Montanide adjuvant) yielding a totalemulsion volume of 2 ml. For subsequent immunizations antigen from 500mosquitoes was similarly emulsified in a total volume of 2,000 μl.Therefore half the concentration of antigen was injected in subsequentimmunizations in the same emulsion volume as the primary inoculum.Control rabbits in all immunizations received 2 ml of emulsioncontaining a 300:700 volume ratio of PBS: adjuvant. All emulsions wereinoculated into rabbits within one hour of emulsification.

Experimental rabbits received 2,000 μl of salivary glands: adjuvant(300:700 ratio by volume) emulsion injected subcutaneously at eightsites, 250 μl per site. Control rabbits received 2,000 μl of PBS:adjuvant (300:700 ratio by volume) emulsion injected subcutaneously ateight sites, 250 μl per site.

v) Rabbit Immunization Schedule

Table 1 provides the immunization schedule used to generate thepolyclonal antibody used in the examples provided herein. This scheduleis not intended to be limiting with regard to the method of polyclonalantibody preparation, but rather is provided only as an example anddisclosure of antibody used herein

TABLE 1 IMMUNIZATION SCHEDULE Time (days) Procedure 0 Pre-bled tworabbits (one control and one experimental) for serum to be used as areference to evaluate antibody titers. 0 First inoculation ofexperimental rabbit with 2,000 μl emulsion of salivary gland materialdissected from 1,000 mosquitoes and control rabbit with 2,000 μlemulsion of 1X PBS and Montanide ISA 720. 21 Second inoculation ofexperimental rabbit with 2,000 μl emulsion of salivary gland materialdissected from 500 mosquitoes and control rabbit with 2,000 μl emulsionof 1X PBS and Montanide ISA 720. 30 Test bled #1; pre-bleed and testsera received for evaluation 42 Third inoculation of experimental rabbitwith 2,000 μl emulsion of salivary gland material dissected from 500mosquitoes and control rabbit with 2,000 μl emulsion of 1X PBS andMontanide ISA 720 51 Test bled #2; sera received for evaluation 57Fourth inoculation of experimental rabbit with 2,000 μl emulsion ofsalivary gland material dissected from 500 mosquitoes and control rabbitwith 2,000 μl emulsion of 1X PBS and Montanide ISA 720. 66 Test bled #3; sera received for evaluation. 92 Based on results of test bleed #3 anadditional inoculation of salivary gland material was administered.Fifth inoculation of experimental rabbit with 2,000 μl emulsion ofsalivary gland material dissected from 500 mosquitoes and control rabbitwith 2,000 μl emulsion of 1x PBS and Montanide ISA 720. 102 Rabbits wereexsanguinated and 45 ml serum was obtained from each rabbit.

B—SGM Elisa Assay

An enzyme-linked immunosorbent assay (ELISA) was used to quantifyattendant material. As an example, salivary gland material (SGM)originating from A. stephensi salivary glands in preparations of P.falciparum sporozoites was quantified. ELISA plates (Nunc MaxiSorp Cert,N/Ster, PS, 96 well Flat-Bottom Immuno Plate, Cat. No. 439454) withdilutions of SGM standards (prepared from salivary glands of uninfectedmosquitoes) and samples of sporozoites from crude salivary glandpreparations as well as samples of the purified sporozoite preparationswere prepared for assay. The primary antibody was rabbit anti-SGM serum(described in A, above) diluted 1:200. Secondary antibody was alkalinephosphatase-conjugated anti-Rabbit IgG diluted 1:5,000 (Promega, Cat.No. S373B). KPL Blue Phos Substrate and Stop Kits (KPL, Cat No. 50-88-06and 50-89-00) were used. Plates were read on a Molecular DevicesMicroplate Reader at 635 nm.

There are two components to the assay used to quantify SGM in thesporozoite preparations. The first consists of generating a standardcurve with reference SGM of known concentration, and the second consistsof quantifying the amount of SGM present in the pre-purification andpost-purification samples. A fresh standard curve is generated for eachassay using an SGM reference standard (RS-SGM) of known SGMconcentration as capture antigen. To establish the standard curve, aserial dilution of decreasing concentration (2.0 to 0.05 μg/mL) of theRS-SGM was prepared in E-199 with 1% HSA. To assess residual SGM inpre-purification and post-purification sporozoite preparations, thesesamples were also serially diluted in E-199 with 1% HSA. Diluted RS-SGMsamples and sporozoite preparations were further diluted to 0.25% HSAwith 1×PBS and added to the ELISA plate in triplicate. This dilutioninto PBS is necessary because it lowers the content of HSA. HSA acts asa blocking agent and can decrease the sensitivity of the assay. TheSGM-coated ELISA plates were incubated first with the rabbit polyclonalanti-SGM antiserum (see paragraph 100) and then with the enzyme-labeledanti-rabbit antibodies, and finally with an enzyme substrate (seeparagraph 101). In the presence of SGM, the enzyme substrate changescolor and the optical densities (ODs) of the contents of the wellschange. The OD of each sample was determined using the microplatereader. To determine the amount of SGM in the sporozoite preparations,the OD values of these samples and the reference standard were fit witha logistic mixed model that has common asymptotes and slope but separateED50 values for each sample. The relative values of the ED50 were usedin combination with the known (or assigned) concentration of SGM in thereference sample to estimate the SGM concentration in thepre-purification sporozoite and VBP samples. The lower and upper 95%confidence limits were similarly determined for the sporozoitepreparations from the overall data incorporating an estimate ofvariation based on three experimental assays that contained a commonreference standard and test samples. From the numbers of sporozoitesknown to be present in each sporozoite preparation, the ng of SGM/25,000sporozoites was calculated as were the reduction factors and percentreductions of the attendant material.

Table 2 shows the results of 10 production campaigns. “Start” representsmeasured amounts in ng SGM/25,000 sporozoites in crude preparationspooled after trituration of salivary glands dissected from infectedmosquitoes. “Final” represents the corresponding measured amount of SGMin preparations which were purified by the methods provided herein. Thismethodology results in substantial purification of the sporozoites—99.9%or more.

It is recognized that the values arising from experimental measurementsprovided for ng SGM/25,000 sporozoites are based on results from thecurrent assay as described, are not absolute, and may be somewhatdifferent if another assay or method of analysis is used.

TABLE 2 PURIFICATION OF SPOROZOITES ng[SGM]/25,000spz Campaign LowerUpper Reduction Residual % Number Start Final 95% CI 95% CI Factor SGM(%) Reduction 1 1,091.65 1.01 0.74 1.38 1,081 0.09 99.91 2 511.00 0.190.12 0.28 2,689 0.04 99.96 3 836.64 0.23 0.16 0.35 3,637 0.03 99.97 4757.11 0.29 0.20 0.41 2,610 0.04 99.96 5 847.90 0.70 0.51 0.96 1,2110.08 99.92 6 780.66 0.65 0.47 0.90 1,201 0.08 99.92 7 893.88 0.61 0.440.85 1,465 0.07 99.93 8 407.37 0.29 0.19 0.44 1,404 0.07 99.93 9 508.420.35 0.24 0.52 1,452 0.07 99.93 10  719.45 0.67 0.48 0.94 1,073 0.0999.91 Geometric 706.88 0.51 0.34 0.67 1,625 0.07 99.93 Mean^(a) ^(a)Datacomprising columns labeled ‘Start’, ‘Reduction Factor’ and ‘PercentReduction’ are skewed. Therefore geometric means were calculated forthese data. Data in the other columns were treated the same way forconsistency.

Example 2 Purification of Sporozoites in Campaign 3

Sporozoites were purified from triturated salivary gland preparationsusing size exclusion filtration as disclosed. The sporozoites weremaintained throughout the purification process in purification medium.At the end of the purification, samples were removed for visualobservation (microscopic examination), bioburden testing (USP <61>),testing for Mycoplasma and Spiroplasma, in vitro determination of viralcontaminants; and SGM-ELISA assay.

A—Purification of Sporozoites

Preparation of material for purification: The triturated dissectionproduct from between 272 and 356 mosquitoes was received altogether in atube. A sample (5 μl) of the dissection product was removed and setaside at 22° C. for visual observation after pooling (FIG. 1 a). Theremaining dissection product was then diluted to 10 ml with thepurification media. A sample (total of 100 μl) was removed at this pointfor counting (20 μl) and the SGM assay (60 μl). All solutions andsamples were kept at about 22° C. for the duration of the purification.

Purification procedure: Using a peristaltic pump at a flow rate of about4 ml/min (about 140 L/hr/m2) the diluted dissection product was pumpedacross three filters (Filter #1, Filter #2 and Filter #3) connected inseries and captured by dead end filtration on Filter #4. Filter #1, adepth filter, had a pore size of 10 microns (Polygard®-CNOptiscale—Millipore Cat. No. SN1HA47HH3). The pore size of Filter #2, adepth filter, was 0.6 micron (Polygard®-CN Optiscale filter—MilliporeCat. No. SN06A47HH3). Filter #3 had a pore size of 1.2 microns (Isoporemembrane, 47 mm in diameter—Millipore Cat. No. RTTP04700) held in aSwin-Loc filter holder (Whatman Cat. No. 420400). Filtered material wascaptured on Filter #4 in a stirred ultrafiltration cell (Millipore,model 8200) fitted with a Isopore membrane, 90 mm in diameter and a poresize of 0.4 μm (Millipore Cat. No. HTTP09030). A total of 100 mlpurification media for run #1 and a total of 120 ml for runs #2-5 werepumped across the membranes together with the diluted dissectionproduct. This was followed with another 100 ml of purification mediaapplied to Filter #4. When the retentate volume reached about 5-10 ml,it was collected together with washes using purification media in atotal of 35 ml that was transferred to a sterile 35 ml Oak Ridgecentrifuge tube. Another 35 ml wash was collected in a similar fashionand also transferred to a second sterile 35 ml Oak Ridge centrifugetube. The 35 ml Oak Ridge tubes were centrifuged at 16,340 g for fiveminutes, and resuspended for counting, and held at about 22° C.

After counting, the material was resuspended to a concentration of about15-18×10⁶ sporozoites/ml. 150 μl was removed for assessment of SGM andstored frozen at −70° C. prior to performing the SGM-ELISA assay. 20 μlwere removed from the purified PfSPZ preparation and applied to amicroscope slide (FIG. 1 b). Settling of sporozoites was allowed for atleast 5 minutes, up to 30 minutes, and the sporozoites were viewed at200× magnification. A similar procedure was carried out for thepre-purification sample (FIGS. 1 a and 1 b). The specific data for eachrun in the campaign are presented in Table 3 and a microscopic analysis(photomicrograph) of pre- and post-purification samples is presented inFIGS. 1 a and 1 b.

In campaign 3, seven runs using between 272 and 356 mosquitoes per run(2146 mosquitoes total) with yields of purified sporozoites per runranging from 40% to 75% (mean 57%) giving a total of 61.3×10⁶ purifiedsporozoites prior to removal of all samples for in-process and releaseassays. The SGM assay (Example 1—Table 2—Campaign 3) showed that thepurified sporozoites contained 0.23 ng SGM/25,000 sporozoites. The SGMcontent of pooled pre-purification material contained 836.64 ng/25,000sporozoites. In this example there was an overall 3,637 foldpurification of sporozoites in the purification process. The modifiedUSP <61> bioburden assay showed that there were no colony forming units,in other words no microbial contamination.

TABLE 3 Sporozoite Yield - Campaign 3 Start of Purification PfSPZ/ Endof Purification Mosquitoes Mosquito Total PfSPZ Total PfSPZ Yield Run#dissected (×10⁻³) (×10⁻⁶) (×10⁻⁶) (%)* 1 356 36.5 13.0 9.8 75.4 2 33043.9 14.5 9.2 63.4 3 272 57.9 15.8 6.6 41.8 4 298 47.8 14.3 10.6 74.1 5326 65.2 21.3 12.7 59.6 6 274 65.2 17.9 8.1 45.3 7 290 37.1 10.8 4.339.8 TOTAL 2146 107.6 61.3 MEAN 50.5 57.1 *% yield = 100 × (#SPZ atend/#SPZ at start)

Example 3 Purification of Sporozoites in Campaign 6

Sporozoites were purified from triturated salivary gland preparationsusing size exclusion filtration as described in Example 2 above. Thesporozoites were maintained throughout the purification process inpurification medium. Samples were removed for visual observation(microscopic examination), microbiological testing, and SGM-ELISA assayto quantify the amount of SGM in the samples. Samples were prepared andpurified as in Example 2.

In campaign 6, eight runs using between 343 and 395 mosquitoes per run(2997 mosquitoes total) with yields of purified sporozoites per runranging from 51% to 87.5% (mean 72.3%) giving a total of 160.5×10⁶purified sporozoites prior to removal of all samples for in-process andrelease assays (Table 4). The SGM-ELISA assay (Example 1—Table2—Campaign 6) showed that the purified sporozoites contained 0.65 ngSGM/25,000 sporozoites. The SGM content of pooled pre-purificationmaterial contained 780.66 ng/25,000 sporozoites. In this example therewas an overall 1.201-fold purification of sporozoites in thepurification process. The modified USP <61> bioburden assay showed thatthere were no colony forming units, in other words no microbialcontamination. Microscopic analyses (photomicrograph) of pre- andpost-purification samples are presented in FIG. 2.

TABLE 4 Sporozoite Yield Campaign 6 Start of Purification PfSPZ/ End ofPurification Mosquitoes Mosquito Total PfSPZ Total PfSPZ Yield Run#dissected (×10⁻³) (×10⁻⁶) (×10⁻⁶) (%)* 1 372 89.7 33.4 29.2 87.4 2 395115.2 45.5 23.2 51.0 3 395 101.3 40.0 22.2 55.5 4 384 99.9 38.4 20.653.6 5 366 72.4 26.5 14.8 55.8 6 343 43.4 14.9 10.1 67.8 7 363 86.8 31.519.3 61.3 8 379 67.3 25.5 21.1 82.7 TOTAL 2997 255.7 160.5 MEAN 84.564.4 *% yield = 100 × (#SPZ at end/#SPZ at start)

Example 4 Infection of a Human Hepatocyte Cell Line (HC-04 [1F9]) withIrradiated (150 GY) P. falciparum Sporozoites from Campaigns 5, 6 and 7

Methods. PfSPZ were incubated for three days with HC-04 (1F9) cells, andthen assessed for expression of PfLSA-1.

TABLE 5 PfLSA-1 expression in liver stage parasites after irradiation ofPfSPZ. Primary Number of Parasites PfSPZ/ Antibody Days in ExpressingPfLSA-1 Campaign well Against Culture Well 1 Well 2 Well 3 Mean/wellSTDEV 5 25,000 PfLSA-1 3 356 327 387 356.7 30.01 6 25,000 PfLSA-1 3 401359 378 379.3 21.03 7 25,000 PfLSA-1 3 426 369 381 392.0 30.05

Results and Interpretation. Detection of Plasmodium falciparum liverstage antigen-1 (PfLSA-1), expressed in liver stage parasites, is anindication of the potency of attenuated and purified sporozoites. Asshown in Table 5, PfLSA-1 is expressed by P. falciparum liver stageparasites after the sporozoites have been exposed to 150 Gy.

Example 5 Infection of a Human Hepatocyte Cell Line (HC-04 [1F9]) withIrradiated (100, 120, 142.5, 150 Gy) or Non-Irradiated P. falciparumSporozoites

Methods. Sporozoites were incubated for three days in HC-04 (1F9) cells,and then assessed for expression of PfLSA-1.

TABLE 6 Number of liver stage parasites expressing PfLSA-1 afterdifferent irradiation doses. Number of Parasites Expressing RadiationPfLSA-1 Dose per Well STD % (Gy) Well 1 Well 2 Well 3 Mean DEV CV 0 363322 341 342.0 20.52 1.37 100 321 302 287 303.3 17.04 1.21 120 286 307323 305.3 18.56 1.22 142.5 318 337 293 316.0 22.07 1.26 150 341 309 285311.7 28.10 1.25

Results and Interpretation. 25,000 PfSPZ, which had been exposed todifferent radiation doses as indicated (Table 6), were added to eachwell of 8-well Lab-Tek slides containing HC-04 (1F9) cells. The slideswere incubated for 3 days and assessed for parasites expressing PfLSA-1.As shown in Table 6, compared to non-irradiated Pf sporozoites, Pfsporozoites that were exposed to 150 Gy had 91% of the activity ofnon-irradiated Pf sporozoites in this assay. This difference did notreach the level of statistical significance in a two-tailed Student'st-Test (p=0.21).

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only. Furthermore, in the foregoing,the present invention has been described with reference to suitableembodiments, but these embodiments are only for purposes ofunderstanding the invention. Various alterations or modifications arepossible so long as the present invention does not deviate from theclaims, which provide a true scope and spirit of the invention.

What is claimed is:
 1. A method of conferring protective immunity in ahuman host against malaria caused by a Plasmodium-species parasite, saidmethod comprising administering to said human host at least one dose ofan aseptic, purified preparation of metabolically active, attenuatedPlasmodium sporozoites selected from the group consisting of P.falciparum, P. vivax, P. malariae, P. ovale, P. knowlesi, and anycombination thereof, said aseptic, purified preparation suitable forpharmaceutical use and comprising less than 85 nanograms of attendantmaterial per 25,000 sporozoites, wherein protective immunity isconferred in said human host.
 2. The method of claim 1 comprisingadministering at least 3 doses of said aseptic, purified preparation. 3.The method of claim 1 comprising administering 5 or more doses of saidaseptic, purified preparation.
 4. The method of claim 1 wherein saidattenuation is established in said aseptic, purified preparation ofmetabolically active, attenuated Plasmodium sporozoites by exposure of aPlasmodium sporozoite to radiation, mutagenic chemicals, metabolicallyinhibiting chemicals, adverse environmental conditions, or byintroducing a heritable genetic alteration, deletion, or mutation in thegenome of said Plasmodium sporozoite.
 5. The method of claim 4 whereinsaid attenuation is established by exposure of said Plasmodiumsporozoite to radiation.
 6. The method of claim 5 wherein saidattenuation is established by exposure of said Plasmodium sporozoite toat least 120 Gy of radiation but no more than 200 Gy of radiation. 7.The method of claim 6 where said attenuation is established by exposureof said Plasmodium sporozoite to 150 Gy of radiation.
 8. The method ofclaim 1 wherein said species of said aseptic, purified preparation ofmetabolically active, attenuated Plasmodium sporozoites is P.falciparum.
 9. The method of claim 1 wherein said aseptic, purifiedpreparation of metabolically active, attenuated Plasmodium sporozoitesis purified from mosquitoes.
 10. The method of claim 9 wherein saidaseptic, purified preparation of metabolically active, attenuatedPlasmodium sporozoites is purified from a pre-purification preparationof mosquito salivary gland material.
 11. The method of claim 1 whereinsaid aseptic, purified preparation of metabolically active, attenuatedPlasmodium sporozoites comprises less than 15 nanograms of attendantmaterial per 25,000 sporozoites.
 12. The method of claim 1 wherein saidaseptic, purified preparation of metabolically active, attenuatedPlasmodium sporozoites comprises less than 1 nanogram of attendantmaterial per 25,000 sporozoites.
 13. The method of claim 1 wherein saidaseptic, purified preparation of metabolically active, attenuatedPlasmodium sporozoites comprises less than 0.12 nanograms of attendantmaterial per 25,000 sporozoites.
 14. The method of claim 10 wherein atleast 98% of attendant material present in said pre-purificationpreparation is removed.
 15. The method of claim 10 wherein at least99.9% of attendant material present in said pre-purification preparationis removed.
 16. The method of claim 10 wherein at least 99.97% ofattendant material present in said pre-purification preparation isremoved.
 17. The method of claim 1 wherein said aseptic, purifiedpreparation of metabolically active, attenuated sporozoites isadministered by a parenteral route of inoculation, said parenteral routechosen from the group consisting of intravenous, intramuscular,intradermal or subcutaneous inoculation.
 18. The method of claim 17wherein said parenteral route is intravenous.
 19. The method of claim 1wherein each dose of said aseptic, purified preparation of Plasmodiumsporozoites comprises at least 15,000 but no more than 400,000 aseptic,purified, metabolically active, attenuated sporozoites.
 20. The methodof claim 1 wherein said protective immunity provides partial protectionagainst the clinical manifestations, pathology or symptoms of malaria insaid human host after subsequent exposure of said host to pathogenicPlasmodium parasites.
 21. The method of claim 1 wherein said protectiveimmunity provides enhanced protection against the clinicalmanifestations, pathology or symptoms of malaria in said human hostafter subsequent exposure of said host to pathogenic Plasmodiumparasites compared to clinical manifestations, pathology or symptoms inan exposed, non-treated host.
 22. The method of claim 1 wherein saidprotective immunity provides full protection against the clinicalmanifestations, pathology or symptoms of malaria in said human hostafter subsequent exposure of said host to pathogenic Plasmodiumparasites.